Process for purification of pneumocandin

ABSTRACT

The process described herein discloses purification process of a secondary metabolite produced by fermentation route. The process involves selective removal of impurities at various stages of washings, charcoalization followed by crystallization. The product is closely related to class of echinocandins and is found to be potent antifungal compound &amp; a key ingredient in the synthesis of antifungal drugs.

FIELD OF INVENTION

The present invention is in relation to purification process of asecondary metabolite produced by fermentation route. The processinvolves selective removal of impurities at various stages of washings,charcoalization followed by crystallization. The product is closelyrelated to class of echinocandins and is found to be potent antifungalcompound & a key ingredient in the synthesis of antifungal drugs.

BACKGROUND OF INVENTION

The instant invention describes a novel process for purification of anaturally occurring secondary metabolite obtained from fermentationroute. The product is closely related to echinocandins and is known tobe a key intermediate to make antifungal agents. In particular,Pneumocandins are discussed in detail here. It is a cyclic hexapeptidewith multiple hydroxyl groups and a hydrophobic dimethylmyristate tailconnected via an amide bond to the alpha amino group of the hydroxylatedomithine residue. According to ] R. E. Schwartz, D. F. Sesin, H. Joshua,K. E. Wilson, A. J. Kempf, K. E. Golden, D. Kuehner, P. Gailliot, C.Gleason, R. White, E. Inamine, G. Bills, P. Salmon, L. Zitano, Pneumocandins from Zalerion arboricola. I. Discovery and isolation, J.Antibiotics 45 (1992) 1853 and G. F. Bills, G. Platas, F. Pelaez, P.Masurekar, Reclassification of a pneumocandin-producing anamorph, Glarealozoyensis gen. et sp. nov., previously identified as Zalerionnarboricola, Mycological Research 102 (1998), Pneumocandin—B₀ can beproduced by fermentation of Glarea lozoyensis (Zalerion arboricola).According to O. D. Hensens, J. M. Liesch, D. L. Zink, J. L. Smith, C. F.Wichman, R. E. Schwartz, J. Antibiotics 45 (1992) 1875 and A. Adeferati,0. Hensens, E. T. T. Jones, J. Tkacz, J. Anti biotics 45 (1992) 1953,the organism can produce other echinocandins in addition to desiredproduct Pneumocandin B0 including its isomers Pneumocandin A₀ and C₀.Structures of Pneumocandins are shown below.

Structures of Pneumocandin—B₀ and its Isomers

The process followed for the purification of crude Pneumocandin-B₀ andrelated compounds involves solvent-solvent extractions, repeated columnpurifications and crystallizations which are tedious and need to lookinto simple and robust process. The instant process is simple and easeto opeate. The claiming process involves novel purification routeinvolving washings with immiscible solvents or water, removal of UVinactive colored impurities and product selective crystallization.Process—1 has been found to have higher yields, better purity and lowerraw material costs. The instant process inturn results in purity morethan 90%.

STATEMENT OF THE INVENTION

The present invention provides a process for purification ofpneumocandin having one or more polar impurities and one or morenon-polar impurities comprising extraction of product from fermentationbroth using suitable solvent and partially concentrated, washing withimmiscible solvent, charcoalization, concentration and filtration,loading the solids obtained from step (d) in a column with an adsorbent,eluting with suitable solvents, concentration of product rich fractionsand crystallization.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWING

FIG. 1: XRD of Pneumocandin—B₀.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in relation to a process for purification ofpneumocandin having one or more polar impurities and one or morenon-polar impurities comprising;

-   -   a) extraction of product from fermentation broth using suitable        solvent and partially concentrated,    -   b) washing with immiscible solvent,    -   c) charcoalization,    -   d) concentration and filtration,    -   e) loading the solids obtained from step (d) in a column with an        adsorbent,    -   f) eluting with suitable solvents,    -   g) concentration of product rich fractions and    -   h) crystallization.

Pneumocandin is Pneumocandin-B₀.

In yet another embodiment of the present invention, suitable solvent forextraction of product from fermentation broth is selected from a groupcomprising n-butanol, sec-butanol, tertiary-butanol and n-propanol.

In still another embodiment of the present invention, the solvent isn-butanol.

In still another embodiment of the present invention, immiscible solventis selected from a group comprising water, pet-ether and cyclohexane.

In still another embodiment of the present invention, the solvent iswater.

In still another embodiment of the present invention, crystallization iscarried out by solvent-antisolvent method.

In still another embodiment of the present invention, the solvent isselected from a group comprising methanol, n-propanol, iso-propanol,n-butanol, sec-butanol, tertiary-butanol and mixture thereof.

In still another embodiment of the present invention, the anti solventis selected from a group comprising acetone, acetonitrile, pet-ether,cyclohexane, ethyl acetate, water and heptane.

In still another embodiment of the present invention, the solvent isn-butanol.

In still another embodiment of the present invention, the anti solventis acetone.

In still another embodiment of the present invention, adsorbent isselected from a group comprising alumina and silica gel.

In still another embodiment of the present invention, the adsorbent isalumina.

In still another embodiment of the present invention, solvent forelution is selected from a group comprising methanol, n-propanol,iso-propanol, n-butanol, sec-butanol, tertiary-butanol, pentane, hexane,heptane, octane, ethyl acetate water and mixture thereof.

In still another embodiment of the present invention, polymorph obtainedis amorphous as shown in FIG. 2.

In still another embodiment of the present invention, wherein purity ismore than 90%.

The invention described herein discloses purification process of thecyclic lipopeptide type molecules for example echinocandin family havingone or more polar impurities and one or more non-polar impurities,comprising following steps:

-   -   a) extraction of product from fermentation broth using suitable        solvent and partially concentrated,    -   b) washing with immiscible solvent,    -   c) charcoalization,    -   d) concentration and filtration,    -   e) loading the solids obtained from step (d) in to a column        preloaded with an adsorbent,    -   f) eluting with suitable solvents,    -   g) concentration of product rich fractions and    -   h) crystallization.

Process described herein is aimed at purification of naturally occurringsecondary metabolite obtained from fermentation route. The process canbe used in general for cyclo—peptide type molecules for exampleechinocandins. In particular the process aims at purification ofPneumocandin—B₀ which is a key product to obtain caspofungin di-acetate,a known antifungal agent, through synthetic route. The instant Processinvolves selective removal of impurities, where product remains in onesolvent and is treated with immiscible solvents for back wash, furtherit is treated with activated charcoal to remove significant amount of UVinactive colored impurities present from the start. This is followed byselective crystallization of product, while impurities largely remain inmother liquor and product precipitates. The first crystallization of theprocess results in amorphous form of Pneumocandin-B₀ with moderatepurity (75-85%).

Further these crystals involves binding of product along with impuritiesonto a adsorbent. Typically N-alumina is used, but other variants ofalumina or silica can also be used. After this, elution is carried outwith solvent composition which is more selective towards impurities.This purification results in removal of other impurities andparticularly A₀ in this step. This is followed by solvent compositionwhich elutes moderate purity product, where some of other remainingimpurities are removed.

Further, elution is carried out with product selective solvent whichelutes high purity product. These high purity fractions are pooled andconcentrated and crystallized to get high purity Pneumocandin-B₀ insolid form (>90%).

In particular, purification of Pneumocandin—B₀ of structure shown aboveis discussed.

The purification process involves extraction of the product fromfermentation broth using suitable solvent, mostly alcohols viz.propanol, iso butanol, t-butanol, n-butanol. The Pooled extract layer isconcentrated to about 30-50 g/kg stage under vacuum at 45-50° C. Thenext step is to remove polar and various other impurities present inconcentrated extract. This is accomplished using a solvent or solventcomposition which is immiscible. As n-bunaol is present in partiallyconcentrated extracts, water can be taken for back wash. Typically ˜2-3volumes of water w.r.t. concentrated pooled extract is added and ismixed well and allowed for settling. n-butanol layer separates fromaqueous layer along with product while large quantities of impuritiesare retained in aqueous layer. ˜10-35% of purity increase can beobtained depending upon impurities in fermentation broth. Water wash canbe repeated to achieve better possible purity. This n-butanol layer isdiluted by adding additional quantity of n-butanol to make productconcentration ˜5-10 g/kg and treated with activated charcoal. Typically0.5:1-5:1 (w/w) charcoal is used. The suspension is stirred well andfiltered through celite bed followed by bed wash. UV inactive coloredimpurities are adsorbed in activated charcoal which further getsadsorbed on celite bed providing removal of non

UV impurities. Also some UV active impurities gets adsorbed by celitegiving chromatographic purity increase of about 10%. The n-butanolproduct layer is concentrated to about 30 g/kg stage.

The next step of purification involves crystallization. Thecrystallization described herein utilizes controlled addition ofanti-solvent and cooling both. Solvent can be any of among methanol,propanol, iso-propanol, n-butanol, t- butanol, iso-butanol etc. andantisolvent can be any of among acetone, acetonitrile, ethyl acetate,water etc whereas preferably acetone is used.

Concentrated pooled product layer after purification steps as discussedabove, is taken in a jacketed vessel for crystallization. Slow additionof antisolvent is initiated at room temperature. Flow rate is adjustedusing a pump in such a way that 5 volumes of antisolvent is added over 4-6 hours. After 2-3 volumes addition of antisolvent, solution reachesclose to saturation point at RT room temperature. At this moment,cooling is started and temperature is brought down to 0-10° C. whilecontinuing addition of antisolvent. The crystallization step issensitive towards initial product concentration, addition rate ofantisolvent and cooling. Fast addition may yield to precipitate theimpurities too, while slow addition may yield to wet paste instead ofsolid powder. Vacuum filtration is used to separate the solids from themother liquor. Solid product is dried under vacuum at 40° C. for 24hours. Depending upon the input purity of material, successivecrystallization can be done to achieve further purification but it isobserved that purification beyond 75-85% becomes extremely difficult.

Solids obtained from above step with purity 75-85% of Pneumocandin—B₀co-exists with its isomers A₀ and C₀ and other closely relatedimpurities. Separation of these impurities is difficult using simpleunit operations like crystallization. Repetitive crystallization offersa very slight increase in purity at a significant loss of product.Conventionally it is required to have chromatographic preparative runsto isolate the B₀. Also, it is known that only normal phasechromatography with very specific mobile phase gives resolution betweenA₀, B₀, C₀ and other related impurities. The instant

Process further involves novel method to further purification to obtainhigh purity (greater than 90%) of Pneumocandin B₀. The process involvesloading of moderate purity (˜75-80%) Pneumocandin—B₀ on an adsorbent.Adsorbent can be chosen among variants of Alumina or silica gel. Neutralalumina is particularly discussed in detail here. At this step, productalong with impurities binds to the adsorbent bed. After this, selectiveremoval of impurities is carried out by varying composition of thesolvents. First solvent composition selective towards impurities ischosen to selectively elute impurities rich fractions, and thencomposition selective towards product is chosen to selectively elutehigh purity product fractions.

Solvents for the elution choosed among methanol, n-propanol,iso-propanol, n-butanol, t-butanol, sec-butanol, ethyl acetate, hexane,heptane and water etc. The study showed that water-rich eluent is highlyselective for related impurities. It was observed that water rich eluentwith small quantities of methanol is more selective towards A₀ isomerand iso-propanol rich solvent with hexane, (typically 80/20 v/v); ismore selective towards C₀. Though it was found that certain compositionof ethyl acetate, methanol water was more selective towards relatedimpurities.

Different ratios of N-alumina with respect to product were tried and30:1 (w_(Alumina)/w_(product)) ratio was found to be optimum for betteryields and purity. Higher ratio of alumina requires more quantity ofsolvents to elute. The same percentage of product as in case of 30:1.Lower ratios of N-alumina result into loss of product.

To start with, N-alumina (30:1 w/w) is packed in a glass column to makea uniform bed. At production scale, nutsche filter can be used for bedpacking. crystals obtained from previous step is dissolved in methanolto make product concentration ˜15-40 g/L. This is loaded on theN-alumina bed. Small amount of flow through is obtained which shows only1-2% of product loaded.

After this, selective elution of impurities is initiated. Typically onecolumn volume of water is passed through the bed which takes out mainlyrelated impurities and some A₀. This is followed by selective elution ofother impurities. Typically gradient mixture of methanol-water is usedfor 5-8 column-volumes which takes almost all of related impurities anda greater extent of A₀. Finally product—B₀ with moderate purity(˜80-84%) elutes and is collected separately. After selective elution ofthese impurities, 100% methanol is used for elution. It is mostselective towards product as almost all related impurities andsignificant amount of A₀ is already eluted, product starts eluting withvery high purity. 10-15 such fractions are eluted and collectedseparately. The purity of initial fractions ranges between 88-90%, whilelater fractions purity varies between 90-95%. These high purityfractions are pooled and concentrated to about 60-100 g/kg stage.Further this concentrate is crystallized using slow addition of acetoneas antisolvent at temperature 0-10° C. as discussed in earlier.

The details of method are exemplified with the help of examples givenbelow . However it should not be construed that the scope disclosure islimited to the examples.

EXAMPLES

20 kg of fermentation broth containing about 31 gm product—PneumocandinB₀ was extracted using 8 Kg of n-butonal.

Example-1

853 gm of n-butanol extract layer consisting of 4.3 gm of product atpurity 22.8% was taken and concentrated to 30 g/kg stage. Thisconcentrate was washed with water 1:1 (w/w) basis. n-butanol layerobtained showed 4.1 gm of product with purity 44.9%. This n-butanollayer was pooled with another n-butanol layer (344 gm) with 4.93 gm ofproduct at purity 26.7%. This pooled n-butanol layer was concentrated to˜30 g/kg stage followed by second stage water wash at 3:1 (w/w) basis.The n-butanol layer separated post water wash weighed 252 gm withproduct 8.4 gm (92.8%) at purity 60%. Aqueous layer weighed 923 gm with0.407 gm (4.5%) product only at purity of 2.7%.

35 gm n-butanol layer was taken out of 252 gm n-butanol obtained above.Analysis showed 1.15 gm of product at concentration of ˜33g/kg andpurity ˜60%. 0.5 gm of activated charcoal was added to this and wasstirred for 1 hour. Separately 15 gm of celite was taken and slurry wasmade using n-butanol. Bed of celite was packed on Buchner funnel andcharcoal suspension was loaded on this celite bed so that charcoal alongwith UV-inactive impurities gets adsorbed on the celite surface.Filtrate (140 ml) consisted of 1.1 gm (95%) with purity 70.1%. Furtherbed was given wash with n-butanol (50 ml) which showed 0.078 gm (˜5%) atpurity 68.5%. These two were pooled (190 ml) and used forcrystallization.

Pooled filtrate and bed wash as obtained above (1.15 gm of product atpurity ˜69%) was concentrated to ˜30 g/kg stage i.e. ˜40 ml. Acetone wasadded drop by drop to the n-butanol-product solution. Flow rate ofacetone was kept at 0.66 ml/min. After 3 volumes of addition of acetone,cooling of reaction mass was started. Temperature was startedprecipitating. Further addition of acetone was continued till 320 ml ofacetone was added. Finally product was filtered out. The final productwas brownish white in color. HPLC analysis showed 1.017 gm (88%) ofproduct with purity 78.6%. XRD analysis showed it to be completeamorphous form.

Example -2

To make purification process robust, crystallization at lower purity ofstarting material was studied. Lower purity at crystallization stage mayarise due to presence of greater percentage of impurities at the extractstage or due to improper treatment at washing and/or other purificationsteps. It has been observed in such a case additional crystallizationmay be required.

240 gm of n-butanol extract layer consisting of 4.97 gm of product atpurity of 22.8% was taken. This was concentrated to productconcentration of ˜30g/kg stage and was given a water wash with 1:1 (w/w)basis. N-butanol layer post water wash showed 4.85 gm (97.6%) of productwith purity of 38%. This was diluted to 395 gm with n-butanol to makeproduct concentration ˜10g/kg.

For charcoalization, 0.5:1 (w/w) of charcoal was taken and mixed withproduct for ˜1hr. This was filtered on celite bed using 15:1 (w/w) ofcelite. 95% product was obtained in filtrate and 5% in bed-wash.

Filtrate and bed wash were pooled and crystallized similar to Example-1.Product (solid) obtained after 1^(st) crystallization showed 3.56 gm ofproduct with purity 58.5%. Post 2^(nd) crystallization, 2.84 gm productwas obtained with 67% purity. It required 3^(rd) crystallization toachieve 81.23% purity with final 1.98 gm product.

It has been observed that crystallization is one of the most criticalstep of purification Process-1. Antisolvent addition rate, quantity,introduction of cooling, initial load concentration and purity, natureof impurities all impact the performance and hence the results ofcrystallization. For example, at very high rate of antisolvent, productalong with impurities precipitates, so purification does not happen thatgood. Further at fast antisolvent addition, more non UV-impuritiesprecipitates, which may not be detected in HPLC purity but visibly looksvery dark. Similarly, if cooling is started at very early stage whenproduct is concentrated, precipitation starts very quickly resultinginto similar phenomenon as discussed with faster antisolvent addition.Further, if addition rate is very slow, it does not crystallizes in aproper way and yields in a cake formation or fluffy mass on filtrationinstead of fine powder. It has also been studied that crystallization at0-3° C. gives higher yields as compared to 8-10° C. due to furtherdecreased solubility of product.

Example: Process 2

Product Pneumocandin-B₀ obtained from process-1 was used for furtherpurification to obtain high purity (>90%) of Pneumocandin—B₀. 1.5 gm ofsolid product was taken and was dissolved in methanol such that productconcentration becomes ˜25g/L. Load was analyzed by HPLC and % area ofproduct and impurities were as following:—Impurity T (RRT 0.33): 0.68%,A₀: 5.57%, B₀: 77.99%, Related Impurity—R(RRT 1.05): 4.42%, C₀: 5.97%.

45 gm of N-alumina was taken and bed was packed in a glass column. Beddimensions were—diameter: 4 cm, height: 3.5 cm. Product was loaded ontothis bed and flow through was collected. Flow through showed only 1.3%of the product with purity 76.92%.

Now selective elution of impurities was carried out as shown in FIG. 2.At first, 1 column volume elution with 100% water is carried out. Thistakes mainly impurity T (RRT 0.33) and impurity A₀, while little ofproduct. HPLC analysis showed 5.57% of T, 26.23% of A₀ and 41.5% of B₀,while product loss was only 4.6%. % Area of R and C0 was 1.2% & 2.5%respectively.

This was followed by elution with methanol/water 25/75% (v/v). Typicalcomposition of a fraction was ˜8% T, ˜12% A₀, ˜60% B₀, ˜2% R and ˜4% C₀,while total product loss was only 1.5% in 6 column volumes.

Next one column volume was eluted with methanol-water 50/50% (v/v). Thisfavors elution of other impurities along with the product resulting intomoderate purity of product. Fraction analysis by HPLC showed ˜8% A₀,83.88% B₀ and 3% R, while T and C₀ were almost absent. Product loss inthis fraction was only 0.51%.

Next 15 column volumes were eluted with 100% methanol and were collectedseparately. First fraction had 85.08% purity and second fraction had86.5% purity. Fractions 3-5 had purity of ˜88.3%, fractions 6-10 hadpurity of ˜90-91.8% while 11-14 had 92-93.7% purity. After this,decreasing trend of purity was observed. Fraction 15 showed 91.99%purity.

Pooled fractions 1-14 showed in total 54.6% of the product with purity90.21%. These pooled fractions were concentrated to ˜25 ml andconcentrate was transferred to a jacketed vessel. Acetone was used asantisolvent and crystallization was carried out as described earlier.Final product was filtered and dried under vacuum as described above.The final product was white in color and complete amorphous in form.HPLC analysis showed 91.37% purity of product—Pneumocandin B₀.

Although Process-1 and Process-2 are described for productPneumocandin-B₀, but it is understood that such techniques can be usedfor purification of other molecules obtained from fermentation which aresimilar to lipo-peptides, particularly echinocandins family. The keys tothe invention in Process-1 & Process-2 are selective removal ofimpurities, while product remains in one solvent from start to end.Process-1 has advantages over reported purification processes in termsof avoiding of repetitive back extraction and concentration steps withor without liquid-liquid extraction, which involves higher operating andraw materials cost along with significant losses of product. Process-1discloses controlled crystallization of product which selectivelyprecipitates the product. Process-1 discloses a novel way ofpurification where product remains in only one solvent from start toend, which improves yield, eases recovery of solvents as well as reducesthe raw material costs. It has been found that process-1 has an overallyield of 55-70% (with respect to broth) with final product purity75-82%. Process-2 has an overall yield of 45-55% with final productpurity 90-93%.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1. A process for purification of pneumocandin having one or more polarimpurities and one or more non-polar impurities comprising; a)extraction of product from fermentation broth using suitable solvent andpartially concentrated, b) washing with immiscible solvent, c)charcoalization, d) concentration and filtration, e) loading the solidsobtained from step (d) in a column with an adsorbent, f) eluting withsuitable solvents, g) concentration of product rich fractions and h)crystallization.
 2. The process as claimed in claim 1, whereinPneumocandin is Pneumocandin-B₀.
 3. The process as claimed in claim 1,wherein suitable solvent for extraction of product from fermentationbroth is selected from a group comprising n-butanol, sec-butanol,tertiary-butanol and n-propanol.
 4. The process as claimed in claim 3,wherein the solvent is n-butanol.
 5. The process as claimed in claim 1,wherein immiscible solvent is selected from a group comprising water,pet-ether and cyclohexane.
 6. The process as claimed in claim 5, whereinthe solvent is water.
 7. The process as claimed in claim 1, whereincrystallization is carried out by solvent-antisolvent method.
 8. Theprocess as claimed in claim 7, wherein the solvent is selected from agroup comprising methanol, n-propanol, Iso-propanol, n-butanol,sec-butanol, tertiary-butanol and mixture thereof.
 9. The process asclaimed in claim 7, wherein the anti solvent is selected from a groupcomprising acetone, acetonitrile, pet-ether, cyclohexane, ethyl acetate,water and heptane.
 10. The process as claimed in claim 8, wherein thesolvent is n-butanol.
 11. The process as claimed in claim 9, wherein theanti solvent is acetone.
 12. The process as claimed in claim 1, whereinadsorbent is selected from a group comprising alumina and silica gel.13. The process as claimed in claim 12, wherein the adsorbent isalumina.
 14. The process as claimed in claim 1, wherein solvent forelution is selected from a group comprising methanol, n-propanol,iso-propanol, n-butanol, sec-butanol, tertiary-butanol, pentane, hexane,heptane, octane, ethyl acetate water and mixture thereof.
 15. Theprocess as claimed in claim 1, wherein polymorph obtained is amorphousas shown in FIG.
 1. 16. The process as claimed in claim 1, whereinpurity is more than 90%.